Multiparametric analyses of cells provide an approach for the simultaneous determination of the activation states of a plurality of cellular components. The activation status of the plurality of cellular components can be measured after exposure of cells to extracellular modulators and in so doing allows the signaling capacity of signaling networks to be determined when compared to the activation status of those networks in the absence of such modulators. The induced activation status of a protein rather than the frequently measured basal phosphorylation state of a protein has been shown in several studies to be more informative, as it takes into account (and reveals) signaling deregulation that is the consequence of numerous cytogenetic, epigenetic and molecular changes characteristic of transformed cells. For example, multiparameter flow cytometry at the single cell level can measure the activation status of multiple intracellular signaling proteins and can assign activation states of these molecules to the varied cell sub-sets within complex primary cell populations.
However, usually multiparametric analyses of cells, e.g., multiparametric flow cytometry, require the use of multiple reagents at precise concentrations to produce robust and reproducible results. Since these data can be used as tools to inform clinical decisions, as well as therapeutic development, it would be beneficial to provide kits comprised of components relevant to a particular application with accompanying relevant usage information.
Protein phosphorylation is a critical post translational process in controlling many cell functions such as migration, apoptosis, proliferation and differentiation. Site specific phosphorylation of proteins can be detected, for example, by incubating cells with fluorochrome-conjugated phospho-specific antibodies using flow cytometry. However, only reagents whose parameters (including but not limited to, concentration, kinetics, fluorochrome to protein ratio) have been optimized can be used to generate robust and reproducible data that can be applied to a specific purpose. Kits comprising two or more reagents recognizing intracellular markers and/or extracellular markers along with an appropriate modulator or modulators to evoke a signaling response appropriate for the signal transduction pathway, specific cell type, disease state, or cellular function can save the end user from the tedious and often costly process of selecting, optimizing and standardizing reagents thereby providing the user with a more streamlined and cost-saving approach for profiling cellular networks in single cells.
It is therefore an objective of the present invention to provide kits that meet such demands.